7-Substituted 8-aza-7-deazaadenosines for modification of the siRNA major groove.

Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, USA.
Organic & Biomolecular Chemistry (Impact Factor: 3.49). 07/2012; 10(32):6491-7. DOI: 10.1039/c2ob25647a
Source: PubMed

ABSTRACT Here we describe the synthesis of new 7-substituted 8-aza-7-deazaadenosine ribonucleoside phosphoramidites and their use in generating major groove-modified duplex RNAs. A 7-ethynyl analog leads to further structural diversification of the RNA via post-automated RNA synthesis azide-alkyne cycloaddition reactions. In addition, we report preliminary studies on the effects of eight different purine 7-position modifications on RNA duplex stability and pairing specificity. Finally, the effect on RNAi activity of this type of modification at eight different positions in an siRNA guide strand has been explored. Analogs were identified with large 7-position substituents that maintain adenosine pairing specificity and are well-tolerated at specific positions in an siRNA guide strand.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Immune stimulation triggered by siRNAs is one of the major challenges in the development of safe RNAi-based therapeutics. Within an immunostimulatory siRNA sequence, this hurdle is commonly addressed by using ribose modifications (e.g., 2'-OMe or 2'-F), which results in decreased cytokine production. However, as immune stimulation by siRNAs is a sequence-dependent phenomenon, recognition of the nucleobases by the trigger receptor(s) is also likely. Here, we use the recently published crystal structures of Toll-like receptor 8 (TLR8) bound to small-molecule agonists to generate computational models for ribonucleotide binding by this immune receptor. Our modeling suggested that modification of either the Watson-Crick or Hoogsteen face of adenosine would disrupt nucleotide/TLR8 interactions. We employed chemical synthesis to alter either the Watson-Crick or Hoogsteen face of adenosine and evaluated the effect of these modifications in an siRNA guide strand by measuring the immunostimulatory and RNA interference properties. For the siRNA guide strand tested, we found that modifying the Watson-Crick face is generally more effective at blocking TNFα production in human peripheral blood mononuclear cells (PBMCs) than modification at the Hoogsteen edge. We also observed that modifications near the 5'-end were more effective at blocking cytokine production than those placed at the 3'-end. This work advances our understanding of how chemical modifications can be used to optimize siRNA performance. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
    ChemBioChem 12/2014; 16(2). DOI:10.1002/cbic.201402551 · 3.06 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Short interfering RNAs (siRNAs) are promising drug candidates for a wide range of targets including those previously considered "undruggable". However, properties associated with the native RNA structure limit drug development and chemical modifications are necessary. Here we describe the structure-guided discovery of functional modifications for the guide strand 5' end using computational screening with the high resolution structure of human Ago2, the key nuclease on the RNA interference pathway. Our results indicate the guide strand 5'-end nucleotide need not engage in Watson-Crick (W/C) H-bonding but must fit the general shape of the 5'-end binding site in MID/PIWI domains of hAgo2 for efficient knockdown. 1,2,3-Triazol-4-yl bases formed from the CuAAC reaction of azides and 1-ethynylribose, which is readily incorporated into RNA via the phosphoramidite, perform well at the guide strand 5'-end. In contrast, purine derivatives with modified Hoogsteen faces or N2 substituents are poor choices for 5'-end modifications. Finally, we identified a 1,2,3-triazol-4-yl base incapable of W/C H-bonding that performs well at guide strand position 12, where base pairing to target was expected to be important. This work expands the repertoire of functional nucleotide analogs for siRNAs.
    Journal of the American Chemical Society 10/2013; 135(45). DOI:10.1021/ja4079754 · 11.44 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ribonucleoside analogs bearing terminal alkynes, including 7-ethynyl-8-aza-7-deazaadenosine (7-EAA), are useful for RNA modification applications. However, while alkyne- and triazole-bearing ribonucleosides are in wide spread use, very little information is available on the impact of these modifications on RNA structure. By solving crystal structures for RNA duplexes containing these analogs, we show that, like adenosine, 7-EAA and a triazole derived from 7-EAA base pair with uridine and are well accommodated within an A-form helix. We show copper-catalyzed azide/alkyne cycloaddition (CuAAC) reactions with 7-EAA are sensitive to the RNA secondary structure context with single stranded sites reacting faster than duplex sites. 7-EAA and its triazole products are recognized in RNA template strands as adenosine by avian myoblastosis virus reverse transcriptase (AMV-RT). In addition, 7-EAA in RNA is a substrate for an active site mutant of the RNA editing adenosine deaminase ADAR2. These studies extend our understanding of the impact of these novel nucleobase analogs and set the stage for their use in probing RNA structure and metabolism.
    ACS Chemical Biology 06/2014; 9(8). DOI:10.1021/cb500270x · 5.36 Impact Factor